The overall goal of this project is to gain further insight into the role that the actin cytoskeleton plays in maintaining homeostasis in the intestinal epithelium. Despite the broad range of cellular processes that are known to require dynamic changes in the actin cytoskeleton, very little is known about the factors that are critical for regulating the rate of actin filament assembly in the highly specialized epithelial cells of the intestine. The Neural Wiskott-Aldrich Syndrome protein (N-WASP) is a major regulator of actin assembly that is ubiquitously expressed, including in the skin and the gut. In order to study its role in epithelial cells, we have recently generated a mouse with gut-specific deletion of N-WASP. Intestinal N-WASP knock-out (iNWKO) mice are born normally and are fertile but are smaller than wild type (WT) controls;they have grossly normal mucosal architecture, with preserved ability of the enterocytes to differentiate into all intestinal cell lineages, and there is no spontaneous inflammation. These mice however exhibit striking apical cell abnormalities, including shortened and clustered microvilli and morphologically abnormal apical junction complexes and the absence of actin dense terminal structures that normally are in continuity with the tight junctions (TJs). iNWKO mice also exhibit defects in targeting the TJ protein occludin to sites of cell-cell contact. These findings correlate clinically with a spontaneous increase in intestinal permeability and dramatic increase in susceptibility to acute inflammatory insult with dextran sodium sulfate (DSS). Our overall hypothesis is that N-WASP plays a critical role in the dynamic regulation of apical cytoskeletal structures which are essential for intercellular contacts and the regulation of paracellular permeability. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to further our understanding of the mechanism(s) that regulate intestinal epithelial structure and enable it to perform its barrier function in both health and disease